Abstract
Abstract: :
Purpose: Collagen gel contraction mediated by corneal fibroblasts (CFs) is thought to contribute to the maintenance of corneal shape after injury . Given that fibronectin is expressed at sites of corneal stromal injury, we investigated whether fibronectin promotes collagen gel contraction mediated by CFs. Methods: Human CFs were cultured in three–dimensional gels of type I collagen in the presence of unsupplemented Eagle’s minimum essential medium containing test agents. Collagen gel contraction was evaluated by daily measurement of the gel diameter for 5 days. The formation of actin stress fibers and the expression of integrin α5 and ß1 chains were examined by immunofluorescence staining and laser confocal microscopy. Results: Fibronectin promoted CF–mediated collagen gel contraction in a dose– and time–dependent manner. Other extracellular matrix proteins (collagen type IV, laminin) or glycosaminoglycans (hyaluronic acid, chondroitin sulfate A or C, dermatan sulfate, heparan sulfate, keratan sulfate) did not exhibit such an effect. Cytochalasin D, which inhibits rearrangement of the actin cytoskeleton, and an RGD peptide antagonist of the fibronectin receptor each inhibited the stimulatory effect of fibronectin on CF–mediated collagen gel contraction. Fibronectin also induced the formation of actin stress fibers, cell spreading, and the establishment of focal contacts containing integrin α5 and ß1 subunits at the tips of stress fibers. Conclusions: Among various extracellular components of the corneal stroma tested, only fibronectin promoted CF–mediated collagen gel contraction. This effect of fibronectin appeared to be mediated by the formation of actin stress fibers and integrin–containing focal contacts. Fibronectin may thus contribute to the maintenance of corneal shape by CFs.
Keywords: cornea: stroma and keratocytes • wound healing • cornea: basic science